Cellular astronomy is a microscopy-based approach to cytometry that uses low magnification, wide field-of-view imaging of stationary cell samples and phenotypic markers borrowed from flow cytometry to enumerate different cell subpopulations [1-4]. By leveraging the simplicity of low resolution microscopy, many cells can be imaged simultaneously over a large field-of-view while being treated similar to point sources, analogous to flow cytometry [1-3, 5]. With cell astronomy, shifting the cellular analysis paradigm towards low-magnification microscopy holds the potential to lower technology barriers to acquiring high throughput cytometry data in the life science research setting due to the prevalence of fluorescence microscopy systems. The use of cell astronomy could allow for more wide-scale deployment of flow cytometry measurements to those labs that possess fluorescence microscopes but do not possess dedicated flow cytometry instrumentation.
To date, cellular astronomy has used fluorescence labels alone to identify and distinguish cell types [2-4]. There would be significant value in additionally measuring the elastic light scattering parameters inherent to flow cytometry with a cellular astronomy system. In flow cytometry, elastic scattering is used to approximate relative cell size (by low angle forward scattered light) and internal cell complexity (by wide-angle side-scattered light) [6, 7]. These forward and side-scattering measures allow for grouping of cell subtypes in a label-free manner (i.e., without fluorescent labeling) [8-14], avoiding unnecessary sacrifice of the optical wavelength spectrum so that more informative molecular biomarkers can be labeled for fluorescence detection. Maintaining the available fluorescent bandwidth by utilizing scatter-based measurements is especially advantageous for microscopy-based cytometry measurements, as many existing fluorescent microscopes are designed to acquire data from a limited number of fluorescence channels. The ability to mimic these forward and side scatter measurements could further unlock wide field-of-view, low resolution microscopy as a potential alternative for gathering high throughput cytometry data.
An object of the invention is a label-free, wide-field scatter-based imaging modality that collects forward and side scatter surrogate data at low-magnification using highly oblique illumination microscopy (HOIM), effectively demonstrating the scatter measurement capabilities of the cellular astronomy approach disclosed herein.
A further object of the invention is image processing algorithms that estimate size and signal intensity from HOIM images of white blood cells.